Molecular phylogenetics of the Ophiocordyceps sinensis-species complex lineage (Ascomycota, Hypocreales), with the discovery of new species and predictions of species distribution

Ophiocordyceps sinensis is a famous traditional Chinese medicine adapted to the alpine environment of the Qinghai-Tibet Plateau and adjacent regions. Clarification of the species diversity of Ophiocordyceps sinensis and its relatives could expand the traditional medicinal resources and provide insights into the speciation and adaptation. The study is prompted by the discovery of a new species, O. megala, described here from a biodiversity hotspot in the Hengduan Mountains, China. Combined morphological, ecological, and phylogenetic evidence supports its distinctiveness from O. sinensis, O. xuefengensis, and O. macroacicularis. Additionally, based on the phylogenetic construction of Ophiocordyceps, a special clade was focused phylogenetically on the more closely related O. sinensis complex, which was defined as the O. sinensis- species complex lineage. A total of 10 species were currently confirmed in this lineage. We made a comprehensive comparison of the sexual/asexual morphological structures among this species complex, distinguishing their common and distinctive features. Furthermore, using the method of species distribution modelling, we studied the species ocurrences in relation to climatic, edaphic, and altitudinal variables for the eight species in the O. sinensis-species complex, and determined that their potential distribution could extend from the southeastern Qinghai-Tibet Plateau to the Xuefeng Mountains without isolating barrier. Thus, the biodiversity corridor hypothesis was proposed around the O. sinensis-species complex. Our study highlights the phylogeny, species diversity, and suitable distribution of the O. sinensis-species complex lineage, which should have a positive implication for the resource discovery and adaptive evolution of this unique and valuable group. Supplementary Information The online version contains supplementary material available at 10.1186/s43008-023-00131-8.

The majority of species in Ophiocordyceps possess firm, darkly pigmented stromata or subiculum, especially those with Hirsutella asexual morphs while some species produce brightly coloured stromata with Hymenostilbe and Paraisaria asexual morphs.The stromata are usually tough, wiry, fibrous, or pliant.Perithecia are superficial to completely immersed, oblique, or ordinal in arrangement.Ascospores are usually cylindrical, multiseptate, either disarticulating into part spores or remaining intact after discharge (Sung et al. 2007).
Species of Ophiocordyceps are distributed worldwide in forest ecosystems of the tropics and subtropics (Petch 1931;Kobayasi 1941;Tzean et al. 1997;Ban et al. 2015;Luangsa-ard et al. 2018;Wang et al. 2018;Araújo et al. 2014Araújo et al. , 2018Araújo et al. , 2020;;Mongkolsamrit et al. 2019;Zha et al. 2021).Although tropical and subtropical areas have the richest species diversity of Ophiocordyceps, alpine or plateau regions cannot be ignored either.Ophiocordyceps sinensis, the Chinese caterpillar fungus, is pre-eminant as a rare traditional and valuable Chinese medicine, and is endemic to the Qinghai-Tibetan Plateau (QTP) and its surroundings in high altitude cold environment (Winkler 2008;Li et al. 2011;Zhang et al. 2012;Hu et al. 2013;Xia et al. 2017;Dai et al 2020).
Considering the uniqueness and specificity of O. sinensis, the diversity of its relatives may be a valuable key to unlocking new understanding of speciation, adaptation and origin of functional component.What, where, and how to form are undoubtedly of scientific importance.Over the last two decades, we have conducted a large-scale survey of entomopathogenic fungi in the alpine regions of southwestern China, one of the world's biodiversity hotspots (Chen et al. 2013a, b;Dai et al 2020;Wang et al. 2020Wang et al. , 2021;;Dong et al. 2022;Sun et al. 2022).Some specimens with huge sclerotia (parasite on a large moth) with long stromata were collected which proved to be a new taxon closely related to O. sinensis was identified.We present a morphological description and phylogenetic analysis of this new fungus and assess the species diversity and potential distribution of the O. sinensis-species complex lineage.

Fungal isolation and culture
The surface of specimens was rinsed with sterile water, and surface-sterilized with 75% ethanol for 1-3 min.Fresh tissue from internal part of sclerota was transfererd to potato dextroseagar (PDA) and cultured at 20 °C in the dark.After purification, cultures were transferred to PDA slants and stored at 4 °C; isolates were deposited in the Yunnan Fungal Culture Collection (YFCC) of Yunnan University, China.

Optical and scanning electron microscope
Specimens collected in the field were photographed and measured using a stereomicroscope (Olympus SZ61).Cultures on slants were transferred to PDA plates and cultured in an incubator for three weeks at 20 °C.Ccircular agar blocks ca. 5 mm diam from plates were cut out and placed on new PDA plates for morphological examination.
For the morphological description, microscope slide cultures were prepared by placing a small piece of mycelium on a 5 mm diam PDA block overlain by a cover slip.Micro-morphological observations and measurements were made using an Olympus CX40 microscope.
For scanning electron microscopy (SEM), 1 cm wide agar blocks were cut from PDA cultures, fixed with 4% glutaraldehyde at 4 °C overnight, washed three times with a phosphate buffer solution (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.4) three times, for10 min each time.Fixed hyphae and conidia were dehydrated using a 50%, 70%, 90% and 100% alcohol series, with 10 min ateach level; and finally dehydrated with supercritical carbon dioxidet.Tthe samples were placed in SEM stubs,,coated with gold-palladium.Conidia and mucilage were examined with scanning electron microscope (S-3400N, Hitachi, Japan) and photographed.

DNA extraction, PCR amplification and sequencing
The genomic DNA of the fungus and its host were extracted with a Fungi DNA isolation Kit according to the manufacturer's instructions (TransGen Biotech, Beijing, China) from the stroma and the surface of sclerotium sections respectively.Genomic DNA was also extracted from the fungal pure cultures (0.05-0.1 g axenic mycelia).The genomic DNA (> 20 ng/μL) was used as the template to amplify DNA fragment.
Six nuclear loci of the fungus were amplified and sequenced, including the internal transcribed spacer (ITS), small and large subunit ribosomal RNAs (nrSSU, nrLSU), transcription elongationfactor-1 alpha (tef), and the largest and second largest subunits of RNA polymerase II (rpb1 and rpb2).The mitochondrial cytochrome coxidase subunit I (cox1) sequences of the insect hosts were also amplified and sequenced.The polymerase chain reaction (PCR) assay was conducted using the manufacturer's manual.The primer information used is provided in Additional file 1: Table S1.PCR products were sequenced on the ABI3700 automatic sequence analyzer (Sangong, Shanghai).The sequences were newly added from seven species and their host insects: Ophiocordyceps megala, O. sinensis, O. laojunshanensis, O. lanpingensi, O. nujiangensis, O. xuefengensis,and O. liangshanensis.

Molecular phylogeny
To construct a phylogenetic tree for O. megala and related species, and recongnize the diversity of the O. sinensis-species complex, representative taxa with five loci (nrSSU, nrLSU, tef, rpb1 and rpb2) were collected from previously published phylogenetic studies of the genus (Sung et al. 2007;Quandt et al. 2014;Sanjuan et al. 2015;Ban et al. 2015, Simmons et al. 2015;Mongkolsamrit et al. 2019).Five loci of each sample were retrieved from GenBank.
A 5-locus dataset was established combining preiously published data with their new sequences generated for the present study.A total of 185 taxa with 5-locus sequence data were selected to represent the diversity of Ophiocordyceps (Table 1).Three Tolypocladium species were chosen as the out groups (Kepler et al. 2014).The ITS region was used to compare the phylogenetic difference among the O. sinensis-species complex.
Sequence alignment of the nrSSU, and nrLSU regions was individually conducted using MAFFT (Katoh et al. 2002).The triplet codon style was set when aligned the exon regions of tef, rpb1 and rpb2,.ensuring that the sequence can be translate to a protein sequence.The alignments were checked visually and adjusted manually where required.Alignment lengths were 4515 bp, 1109 for nrSSU, 1026 for nrLSU, 931 for tef, 553 for rpb1, and 935 for rpb2.All five loci were combined into a single dataset and 11 data partitions were defined: one each for nrSSU and nrLSU plus nine for each of the three codon positions for the protein coding genes tef, rpb1 and rpb2.
A Bayesian Inference phylogenetic tree was inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under partition model (2 parallel runs, 50,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in.The operation stop rule was set when the average standard deviation of split frequencies was below 0.01.The convergence of the runs was checked using Tracer v.1.6(Rambaut et al. 2014).Due to the huge amount of data and the time-consuming process, we used the online platform (CIPRES, https:// www.phylo.org/ porta l2/) to complete the calculations.The contree file was visualized with FigTree v.1.6(http:// tree.bio.ed.ac.uk/ softw are/ figtr ee/).
In addition, the ITS sequences were used to clarify the phylogenetic relationships among the O. sinensis-complex.The cox1 sequences were used to identify their host insects.The whole process of these two datasets were conducted in PhyloSuite (Zhang et al. 2020).

Species distribution modelling
Species occurrence data were mainly collected from our ongoing field studies.Bioclim variables were downloaded from the CliMond Archive (https:// www.climo nd.org/) (Kriticos et al. 2012).
A total of 32 species occurrence data were collected (Table 2).And a total of 35 typical climate, edaphic and altitudinal variables at a grid resolution of 10' were obtained from the CRU CL2.0 dataset.These factors contain the core set of 19 variables (temperature and precipitation), and an extended set of 16 solar radiation and soil moisture variables at a global extent (Additional file 1: Table S2).All climate variables data sources were from the period 1961-1990 (30 years centred on 1975).
Species distribution modelling was based on species redundance with MaxEnt V3.4.1 (Phillips et al. 2006;Elith et al. 2011).Randomly, 25% of the data points were extracted as the test data, and "do jackknife to measure variable importance" was selected.The output grid  format was set as "cloglog." The it was visualized with Global mapper17 (https:// www.bluem arble geo.com).
In the phylogenetic tree of Ophiocordyceps constructed with 5 genes data, a special clade were focused on that had close phylogenetic relationship with O. sinensis.We defined it as the O. sinensis-species complex lineage (MLBS = 100, BPP = 1.0).
After sufficient integration, we confirmed that the clade currently contains 10 species, including O. megala reported in this study (Fig. 2).Among

Host identification
Hosts of O. megala and others eight O. sinensis-species complex were identified based on the cox1 gene.The ML tree showed that all hosts belonged to Hepialidae (Lepidoptera), but scattered in different phylogenetic clade (Fig. 3).(Fig. 4).

Etymology
After the long and large (massive) stromata and huge host.

Diagnosis
Differs from related species mainly in having massive stromata, long phialides, large single conidia, and the huge-sized host.

Notes
Specimens with mature sexual structures were not found among the many specimens of O. megala used in this study.However, a specimen numbered NTUH 17-004 previously identified as O. macroacicularis had the same ITS sequence characteristics with O. megala (Fig. 2B).Their 28S ribosomal RNA gene fragment (~ 800 bp) (MH461122) was also total the same (Ariyawansa et al 2018).Meanwhile, NTUH 17-004 had significant difference in the stromata, peritheciua and asci megala.Thus, the ascus data could be supplied based on this specimen, it provided extremely important circumstantial evidence for the description of the new species O. megala (Fig. 4S-X).
Based on these characteristics, we illustrated a hypothetical O. megala with sexual structures (Fig. 5).

Species clarification of the O. sinensis-species complexs
Based on the phylogeny, we clarified 10 species in the O. sinensis-species complex.To conduct a more comprehensive sexual/asexual characteristics comparison, the detailed description of the sexual morphs of O. nujiangensis and asexual morphs of O. xuefengensis were supplied in our present study, as this was lacking in the previous papers (Wen et al. 2013;Sun et al. 2022) (Fig. 6).And on this basis, we could summarize some common characteristics of this lineage.

Asexual stage
Hirsutella-like.The colony slow growing on PDA and hard in texture.Mostly brown to dark brown.Conidiogenous cells-arising from hyphae directly or laterally, monophialidic, hyaline, smooth-walled, subulate, tapering gradually into slender neck.Conidia-arising from the apex of the conidiogenous cells, oval or citriform shape, usually single, rare 2(-3) aggregated.
In addition to commonalities, we conducted the list of sexual-asexual morphological comparison among O. sinensis-species complex (Table 3), the differences between traits were quantified, which could be used as species clarification and retrieval.There were great differences among hos, perithecia and asci.Significant differences can also be found in Conidiogenous cells and Conidia.

Suitable distribution of the O. sinensis-species complex
A prediction of the area suitable for eight species in the O. sinensis-species complex was obtained with the species distribution modeling method.Major suitable distribution areas (highlight with red colour) appear in southern and southeastern edge of the Hengduan Mountains, the Yunnan-Guizhou Plateau and local areas of the Xuefeng Mountains, and some suitable areas exist in eastern Taiwan and Fujian Province (Fig. 7).The main geographical distribution, especially in the southwest of China, predominantly present not sporadic but continuous large regions.
A biodiversity corridor hypothesis (生态长廊假说) is deduced for the O. sinensis-species complex based on their potential suitable distribution prediction.The O. sinensis-species complex evidently could have an entirely suitable distribution area from west to east, which could provide an excellent ecological environment for the spread and evolution of this unique group, so that it could form a rich diversity and radiation adaptation characteristics.This ecological corridor mainly starts from the Qinghai-Tibet Plateau in the west and extends to the Xuefeng Mountains in the east, passing through the Hengduan Mountains and the Yunnan-Guizhou plateau (Fig. 7).Furthermore, O. megala is delimited by its long, large, and lignified stroma, and has a huge Endoclita host, with longer phialides and larger conidia, which distinguish it from the other species.Both morphological and phylogenetic analyses (5-locus and ITS sequence data, respectively) show that O. megala is a new species with a Hirsutella-like asexual morph.The discovery of O. megala has further enriched the species diversity in the O. sinensis-species complex.
We also suggest the common local name "ChongCaoWang" (虫草王) in Lanping County as its formal Chinese    name; "Chongcaowang" expresses the huge morphological features.The potentially suitable distributional regions are predicted to extend from the southeastern QTP to the Xuefeng Mountains with non-sporadically fragmented regions.Just as the Hengduan Mountain are hypothesized to be a corridor between the Palaearctic and Oriental regions, bridging the faunas of the north and south (Wu et al. 2017), we also propose that the Hengduan Mountains and Yunnan-Guizhou plateau are the biodiversity corridor for the O. sinensis-species complex.

CONCLUSIONS
In our study, the phylogeny, species diversity and potential suitable distribution are systematically illustrated and discussed of the O. sinensis-species complex lineage.And we described Ophiocordyceps megala new to this lineage.The biodiversity corridor hypothesis is proposed based on the suitable distributions prediction of O. sinensis-species complex.And the high confidence predictions should have positive guiding significance for subsequent resource discovery.The detailed description and comparison of these 10 species also have a positive implication for the adaptive evolution of this important valuable group.As the limited information from the morphology and phylogeny, multi-omics research is very necessary for the variation and adaptation around the O. sinensis-species complex.

Fig. 2
Fig.2The phylogenetic structure of O. sinensis-species complex based on two datasets.A 5-locus of nrSSU, nrLSU, tef, rpb1 and rpb2 datasets; B ITS gene.Notes: the holotype of each species was marked with superior characters T

Fig. 3
Fig. 3 The host identification of of O. sinensis -species complex based on cox1 sequences data

Table 1 (
them, O. laojunshanensis was the nearest to O. sinensis.While O. liangshanensis, O. karstii and O. nujiangensis had closed continued) The sequences of Ophiocordyceps megala were submitted to NMDC (National Microbiology Data Center), and the NMDC accession numbers were list.The others sequencs accession numbers were obtained from GenBank relationships.Our reported O. megala was closely related to O. macroacicularis and O.xuefenensis.By contrast, the location of O.robertsii and O.lanpingensis were less certain, which phylogentic position was significant difference in 5-locus and ITS datasets.

Table 2
The species occurrence data of eight species within O. sinensis -species complex

Table 3
Morphological comparison of ten species among Ophiocordyceps sinensis-species complex